Atomic Force Microscopy (AFM) Analysis of the Bacterial Polar Protein Popz

Structural studies of filament-forming biomolecules have classically relied on optical imaging methods such as fluorescence and electron microscopy. While these methods have yielded novel insights into mechanisms of macromolecular assembly, they often require perturbative labeling or staining procedures that can affect the assembly process. In contrast, Atomic Force Microscopy (AFM) has emerged as a powerful imaging tool to directly study native nanostructures at high resolution without labeling. Unlike optical methods, AFM utilizes a nano-scale cantilever to generate a topography map of surface-immobilized molecules in air or in aqueous environments, allowing studies under more physiological or dynamic conditions. However, like all imaging methods, most biological samples require electrostatic or covalent interaction of the biomolecule with a surface for imaging, and care must be taken to avoid surface-specific effects on the sample. Here, we examined the structural organization of the bacterial polar scaffold protein PopZ by AFM, and analyzed the effect of surface-immobilization conditions on PopZ's nanostructural assembly characteristics. We measured the structures and densities of PopZ complexes on positively and negatively charged as well as hydrophobic surfaces, and compared structural organization of these assemblies in aqueous environments. Our results illustrate how choice of surface immobilization conditions can affect structural studies of polymeric assemblies, and demonstrate the tremendous advantages of AFM for directly imaging biomolecules in aqueous, physiological conditions. Finally, our results provide new insight into the structures of multimeric PopZ nano-assemblies that have been thus far unattainable using standard EM methods, providing direct evidence for PopZ self-assembly into organized three-dimensional polymeric networks.